Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit
Engineering of clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) system has enabled versatile applications of CRISPR beyond targeted DNA cleavage. Combination of nuclease-deactivated Cas9 (dCas9) and transcriptional effector domains allows...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-04-01
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| Series: | Genes |
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| Online Access: | https://www.mdpi.com/2073-4425/14/4/906 |
| _version_ | 1797605311089999872 |
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| author | Brittany Chapman Jeong Hoon Han Hong Jo Lee Isabella Ruud Tae Hyun Kim |
| author_facet | Brittany Chapman Jeong Hoon Han Hong Jo Lee Isabella Ruud Tae Hyun Kim |
| author_sort | Brittany Chapman |
| collection | DOAJ |
| description | Engineering of clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) system has enabled versatile applications of CRISPR beyond targeted DNA cleavage. Combination of nuclease-deactivated Cas9 (dCas9) and transcriptional effector domains allows activation (CRISPRa) or repression (CRISPRi) of target loci. To demonstrate the effectiveness of the CRISPR-mediated transcriptional regulation in chickens, three CRISPRa (VP64, VPR, and p300) and three CRISPRi (dCas9, dCas9-KRAB, and dCas9-KRAB-MeCP2) systems were tested in chicken DF-1 cells. By introducing guide RNAs (gRNAs) targeting near the transcription start site (TSS) of each gene in CRISPRa and CRISPRi effector domain-expressing chicken DF-1 cell lines, significant gene upregulation was induced in dCas9-VPR and dCas9-VP64 cells, while significant downregulation was observed with dCas9 and dCas9-KRAB. We further investigated the effect of gRNA positions across TSS and discovered that the location of gRNA is an important factor for targeted gene regulation. RNA sequencing analysis of <i>IRF7</i> CRISPRa and CRISPRi- DF-1 cells revealed the specificity of CRISPRa and CRISPRi-based targeted transcriptional regulation with minimal off-target effects. These findings suggest that the CRISPRa and CRISPRi toolkits are an effective and adaptable platform for studying the chicken genome by targeted transcriptional modulation. |
| first_indexed | 2024-03-11T04:59:15Z |
| format | Article |
| id | doaj.art-a038da885bfe4045af39c38eb62e3135 |
| institution | Directory Open Access Journal |
| issn | 2073-4425 |
| language | English |
| last_indexed | 2024-03-11T04:59:15Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Genes |
| spelling | doaj.art-a038da885bfe4045af39c38eb62e31352023-11-17T19:24:15ZengMDPI AGGenes2073-44252023-04-0114490610.3390/genes14040906Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi ToolkitBrittany Chapman0Jeong Hoon Han1Hong Jo Lee2Isabella Ruud3Tae Hyun Kim4Department of Animal Science, The Pennsylvania State University, University Park, PA 16802, USADepartment of Animal Science, The Pennsylvania State University, University Park, PA 16802, USADepartment of Animal Science, The Pennsylvania State University, University Park, PA 16802, USADepartment of Animal Science, The Pennsylvania State University, University Park, PA 16802, USADepartment of Animal Science, The Pennsylvania State University, University Park, PA 16802, USAEngineering of clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) system has enabled versatile applications of CRISPR beyond targeted DNA cleavage. Combination of nuclease-deactivated Cas9 (dCas9) and transcriptional effector domains allows activation (CRISPRa) or repression (CRISPRi) of target loci. To demonstrate the effectiveness of the CRISPR-mediated transcriptional regulation in chickens, three CRISPRa (VP64, VPR, and p300) and three CRISPRi (dCas9, dCas9-KRAB, and dCas9-KRAB-MeCP2) systems were tested in chicken DF-1 cells. By introducing guide RNAs (gRNAs) targeting near the transcription start site (TSS) of each gene in CRISPRa and CRISPRi effector domain-expressing chicken DF-1 cell lines, significant gene upregulation was induced in dCas9-VPR and dCas9-VP64 cells, while significant downregulation was observed with dCas9 and dCas9-KRAB. We further investigated the effect of gRNA positions across TSS and discovered that the location of gRNA is an important factor for targeted gene regulation. RNA sequencing analysis of <i>IRF7</i> CRISPRa and CRISPRi- DF-1 cells revealed the specificity of CRISPRa and CRISPRi-based targeted transcriptional regulation with minimal off-target effects. These findings suggest that the CRISPRa and CRISPRi toolkits are an effective and adaptable platform for studying the chicken genome by targeted transcriptional modulation.https://www.mdpi.com/2073-4425/14/4/906activationchickenCRISPRdCas9interference |
| spellingShingle | Brittany Chapman Jeong Hoon Han Hong Jo Lee Isabella Ruud Tae Hyun Kim Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit Genes activation chicken CRISPR dCas9 interference |
| title | Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit |
| title_full | Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit |
| title_fullStr | Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit |
| title_full_unstemmed | Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit |
| title_short | Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit |
| title_sort | targeted modulation of chicken genes in vitro using crispra and crispri toolkit |
| topic | activation chicken CRISPR dCas9 interference |
| url | https://www.mdpi.com/2073-4425/14/4/906 |
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